Abstract 270: The TWIST1-microRNA-22 axis represses estrogen receptor alpha expression in breast cancer

• Published in: Cancer research (Chicago, Ill.) Ill.), 2011-04, Vol.71 (8_Supplement), p.270-270
• Main Authors: Vesuna, Farhad, Lisok, Ala, Domek, John, van Diest, Paul, Raman, Venu
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2011-270

Background: TWIST1 (Twist) is a master regulator of embryonic cell fate specification and differentiation. It mediates its transrepressive and transactivating functions by binding to consensus E-box sequences within promoters. Importantly, ectopic expression of Twist in breast epithelial tissue is an initiator of breast cancer. Estrogen and estrogen receptor (ER) expression play a crucial role in breast cancer. ER expression is regulated by methylation, chromatin inactivation, alternative promoter utilization, mutations, as well as cis and trans-acting elements. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. We show here that Twist binds to E-boxes in the promoter of the microRNA miR-22 and upregulates its expression in breast cancer. Furthermore, the dysregulated miR-22 binds to the 3’-UTR of ER and downregulates its expression leading to an ER negative state. Methods: We isolated miRNA from breast cancer cells and assayed for miR-22 expression by qRT-PCR. A miR-22 construct was transfected into breast cancer cells to force expression of miR-22 and analyze effects on ER. Inhibition studies were carried out using targeted knockdowns of miR-22 and analyzed by qRT-PCR. Fine mapping of the miR-22 genomic structure was performed by RACE. The miR-22 promoter containing three E-boxes was cloned and used in dual luciferase assays to study Twist binding and miR-22 activation. Chromatin immunoprecipitation assays were carried out to confirm Twist binding. The 3’-UTR of the ER was cloned into the pMIR-Report vector and the construct used to study miR-22 binding. Functionality of miR-22 binding to the ER 3’-UTR leading to loss of ER expression was confirmed by immunoblotting. Results: We found that miR-22 was highly expressed in cell lines expressing Twist and that ER expression was inversely proportional to miR-22 in all cancer cell lines. We discovered that miR-22 gene consists of three exons spanning over 4.5 kb in the C17orf91 putative gene in chromosome 17. Forced expression of Twist increased miR-22 expression while knockdown of Twist lowered miR-22 expression in vitro. Consequently, forced expression of Twist and miR-22 lowered expression of ER in MCF-7 as seen at both RNA and protein levels. Mechanistically, both miR-22 binding sites in the ER 3’-UTR were important for miR-22 functionality and removal of either site was sufficient to abrogate ER downregulation. Finally, we observed that miR-22 expression wa